The invention relates to a gene delivery device for localizing and enhancing the efficacy of gene transfer.
Gene transfer is a powerful technique which uses a biological vehicle (such as an engineered adenovirus) to introduce a specific gene of interest into a target tissue. Studies have characterized the morphologic, biochemical, and functional effects of recombinant gene expression in a wide variety of tissues, including animal and human cerebral arteries, and support the applicability of gene therapy for the treatment of vascular diseases, including cerebrovascular disease (Chen, et al., Trends in Pharmacological Sciences 19: 276-286,1998; Khurana, et al., Journal of Cerebral Blood Flow and Metabolism 20: 1360-1371,2000).
Ooboshi, et al. (Circulation Research 77: 7-13,1995) carried out the first gene transfer to cerebral arteries in vivo. In their purely morphologic study, the investigators delivered a replication-incompetent adenoviral vector (expressing recombinant xcex2-galactosidase gene) into the cerebrospinal fluid (CSF) of Sprague-Dawley rats held in various anatomical positions. One to seven days following injection, the transduced brains of the animals were examined histochemically after appropriate staining. The authors reported: 1. Distribution of recombinant protein staining consistent with the anatomical position in which the rat was held; 2. Good transduction of the adventitial layer of large and small cerebral arteries (consistent with perivascular gene delivery); and 3. Undetectable xcex2-galactosidase expression by day seven following injection (i.e., indicative of short-term recombinant gene expression). In the first functional study of transduced intracranial arteries, Chen, et al. (Circulation Research 80: 327-25 335,1997) reported the morphologic, biochemical, and vasomotor effects of ex vivo transduction of canine basilar artery with an adenoviral (Ad) vector expressing recombinant endothelial nitric oxide synthase (eNOS). Their principal findings were: 1. Recombinant protein was expressed mainly in the adventitia and, to a lesser extent, in the endothelium of transduced arteries (consistent with ex vivo transduction); 2. Expression of recombinant eNOS in the arterial wall was associated with beneficial vasomotor effects including significantly enhanced relaxations to calcium ionophore A23187, a compound whose receptor-independent relaxing actions are nitric oxide (NO)-mediated, and reduced contractions to uridine triphosphate; and 3. Basal production of cyclic 3xe2x80x25xe2x80x2-guanosine monophosphate (cGMP; the second messenger for NO-mediated signaling) was significantly increased in AdeNOS-transduced arteries. Immediately following this study, similar findings were reported by Chen, et al. (Proceedings of the National Academy of Sciences of the United States of America (PNAS) 94: 12568-12573,1997) in vivo in dogs. Together, these studies indicated that cerebral arterial tone could be favorably modulated by recombinant eNOS expression in the vessel wall, i.e., that gene transfer could achieve a therapeutic effect. That these findings are reproducible in nonpostmortem human cerebral arteries has been recently demonstrated by Khurana, et al. (supra).
To date, most ex vivo and in vivo gene transfer studies in the cardiovascular system have utilized recombinant adenoviruses in the titer range of 109-1010 plaque forming units (PFU), exposing tissues to approximately 1 to 10 billion infectious (viral) units. Based on studies related to cerebrovascular gene transfer, this translates to exposing each target cell to approximately 1000 infectious units, thereby setting the stage for excessive immunogenicity and cytotoxicity from the relatively large xe2x80x9cvector load.xe2x80x9d Despite the large amounts of virus being delivered to tissue sites, experiments involving recombinant xcex2-galactosidase- or luciferase-based quantification of adenovirus-mediated gene transfer efficiency demonstrate relatively poor transduction of arteries ex vivo, which is likely to be even poorer in vivo (Heistad, et al., Stroke 27: 1688-1693,1996). To some extent this phenomenon may be attributable to a relative paucity of coxsackie virus-adenovirus receptor (CAR), in cerebral arteries (Heistad, et al., supra). However, regardless of the underlying reason(s), development of techniques to greatly reduce the number of infectious units delivered to tissues, including blood vessels, ex vivo and ultimately in vivo, is required in order to reduce the likelihood and severity of an adverse response to the vector due to the sheer number of particles delivered to the host.
Several recent publications have reported the feasibility of direct gene transfer, without the use of viral vectors, into tissues such as muscle (Ferry, et al., PNAS 88: 8777-8781,1991; Quantin, et al., PNAS 89: 2581-2584,1992), hematopoletic stem cells (Clapp, et al., Blood 78: 1132-1139,1991), arterial wall (Nabel, et al., Science 2: 1342-1344,1989), nerve (Price, et al., PNAS 84: 156-160,1987), and lung (Rosenfeld, et al., Science 252: 431-434,1991). Direct injection of DNA into skeletal muscle, (Wolff, et al., Science 247: 1465-1468,1990) and heart (Kitsis, et al., PNAS 88: 4138-4142,1991), and injection of DNA-lipid complexes into the vasculature (Lim, et al., Circulation 83: 2007-2011,1991; Leclerc, et al., Journal of Clinical Investigation 90: 936-944,1992; Chapman, et al., Circulation Research 71: 27-33,1992) has also been reported to yield a detectable level of recombinant gene-product expression in vivo. However, conventional vector delivery methods, including ex vivo xe2x80x9cdrippingxe2x80x9d or xe2x80x9cimmersion techniques, and in vivo dripping or injection, remain inefficient and poorly tissue-specific.
Heistad and colleagues (supra) first reported the use of a mechanical method, namely controlled animal head-tilt, to assist in localizing vectors injected into the CSF via the cisterna magna to arteries in the circle of Willis. While this technique is helpful, it remains relatively nonspecific and operator-dependent. A molecular targeting technique using a cell-specific promoter such as SM22xcex1 (selective for smooth muscle cells) rather than a cell-nonspecific promoter such as that derived from cytomegalovirus (CMV) has been demonstrated to be effective in vitro (Kim, et al., Journal of Clinical Investigation 100: 1006-1014,1997), and may be useful in vivo to selectively target vascular as opposed to neuronal or glial tissue. However, at present, there is no way to reliably distinguish between smooth muscle cells in different cerebral arteries, and therefore the problem of being able to target specific vascular territories remains unsolved using this approach.
The invention provides a device and method for increasing the efficiency of gene transfer by localizing a vector at a desired tissue site and by increasing the uptake of the vector by cells at the tissue site. In one embodiment, the invention provides a method for delivering a pharmaceutical composition comprising a nucleic acid to a tissue site. The method comprises the steps of providing a gene delivery device comprising a contact surface, and applying the pharmaceutical composition to the contact surface. The contact surface is then contacted to the tissue, thereby placing and localizing the pharmaceutical composition at the tissue site. Contact with the tissue by the contact surface significantly enhances transduction of the tissue by the nucleic acid relative to transduction of noncontacted tissue to which the pharmaceutical composition is applied. In one embodiment of the invention, transduction efficiency is enhanced greater than 10-fold.
In one embodiment of the invention, the pharmaceutical composition comprises a nucleic acid selected from the group consisting of DNA, RNA, anti-sense molecules, triple-helix-forming nucleic acids, aptamers, and ribozymes. In another embodiment of the invention, the nucleic acid is encapsulated, such as by viral proteins or by a liposome coat. In a further embodiment of the invention, the nucleic acid is an adenoviral vector encapsulated by adenoviral glycoproteins, and transduction of cells at the tissue site includes infection by the adenovirus. In still a further embodiment of the invention, the nucleic acid is bound to or associated with a targeting molecule which binds to a cell at the tissue site.
In one embodiment of the invention, the pharmaceutical composition is placed at the tissue site along with a polymer compound which coats the tissue site. In one embodiment of the invention, the polymer compound is a tissue glue (e.g., fibrin glue); in another embodiment of the invention, the polymer compound is a hydrogel.
In one embodiment of the invention, the contacting is performed by moving the contact surface across the tissue site, such as by a back and forth and/or circular motion. In one embodiment, the contacting compresses tissue at the tissue site relative to noncontacted tissue, while in another embodiment, the contacting causes a portion of the tissue site to temporarily lie over another portion of the tissue site. In still another embodiment of the invention, cells at the tissue site are abraded in the process of contacting.
In one embodiment of the invention, tissue sites include, but are not limited to, the outer or inner surface of a blood vessel, skin, wounded tissue, mucosa, the outer or inner surface of an abdominal or thoracic or special sensory organ, the cortical or ventricular surface or parenchyma of the brain, the spinal cord or its surrounding tissue, meningeal tissue, a muscle, tendon, cartilage, joint, or bone. In one embodiment, the tissue site is cerebrovascular tissue. In another embodiment, the tissue site is cardiovascular tissue.
In one embodiment of the invention, the tissue site is contacted with the contact surface through an open surgical field. In another embodiment of the invention, the contact surface is first inserted into the lumen of an organ or a vessel prior to contacting a tissue site, such as by using a medical access device, such as a catheter or endoscope. In one embodiment of the invention, the contact surface is part of a gene delivery device, at least a portion of which is radiopaque.
In still another embodiment of the invention, the pharmaceutical composition comprises a solution which comprises detectable moieties, and placement and localization of the pharmaceutical composition at the tissue site is monitored by detecting the detectable moieties. In one embodiment of the invention, the solution comprises green-fluorescent protein (GFP). In another embodiment of the invention, the solution itself is radiopaque. In yet another embodiment of the invention, the solution contains a dye visualizable by the naked eye.
In still a further embodiment of the invention, the contact surface is in communication with an optical system including a light source, a light-transmitting element, one end of which is in proximity to the contact surface, and a detector. In this embodiment, contacting of the contact surface with the tissue site is monitored by detecting light transmitted from the light source through the transmitting element. In the embodiment of the invention where the pharmaceutical composition comprises a solution which comprises detectable moieties, placement and localization of the pharmaceutical composition can also be monitored. In one embodiment of the invention, the compression or folding of tissue is monitored. In still another embodiment, the placing and/or uptake of the pharmaceutical composition is monitored. In a further embodiment of the invention, the monitoring of the compression or folding of tissue and/or of the placing and/or uptake of the pharmaceutical composition is used to determine whether further contacting is necessary. In still a further embodiment, the medical access device comprises a cutting element, and a tissue site is exposed to the contact surface by the cutting element, prior to contacting with the contact surface. In one embodiment, the cutting element is a laser.
In one embodiment of the invention, the contact surface comprises a plurality of contact elements, such as bristles, fibers, the protrusions of a sponge, prongs, tines, and the like. In another embodiment of the invention, the contact elements are the bristles of a paintbrush. In one embodiment of the invention, a gene delivery device comprising a contact surface is used to contact the tissue and to deliver the pharmaceutical composition. In still another embodiment of the invention, the contact surface is a surgeon""s gloved finger.
In one embodiment of the invention, the gene delivery device comprises a lumen with an opening in proximity to the contact surface, and the pharmaceutical composition is delivered to the contact surface through the lumen. In another embodiment of the invention, the pharmaceutical composition further comprises a polymerizable compound which polymerizes when the contact surface is contacted to the tissue site. In still another embodiment of the invention, the lumen is divided into a first and second channel sharing a common wall and the pharmaceutical composition and polymerizable compound are delivered through the first channel while a polymerizing agent is delivered through the second channel. When the polymerizable composition and pharmaceutical composition and polymerizing agent come into contact with each other at the tissue site, the polymerizable composition polymerizes, further localizing the pharmaceutical composition at the tissue site.
In one embodiment of the invention, a gene delivery device for use in performing the method is provided which comprises a shaft coupled to a contact surface. In one embodiment, the shaft comprises a shaft housing having a first end and a second end and defining a lumen. The first end comprises an opening and is in communication with a contact surface for contacting a tissue. However, in another embodiment, the first end comprises a plurality of openings.
In one embodiment of the invention, the contact surface comprises a plurality of elongated contact elements, each contact element comprising a base and a distal tip, the base being joined to the first end of the shaft housing and the distal tips separate from each other. In one embodiment, each contact element is a bristle, a fiber, tine, or a prong. In one embodiment of the invention, the plurality of contact elements at least partially surround the opening. In a further embodiment, the contact elements comprise a longitudinal axis which is at a less than 180 degree angle with respect to the longitudinal axis of the shaft housing.
In one embodiment of the invention, the contact surface is a porous material, such as a sponge, which is fixed to the first end of the shaft housing. In another embodiment, the porous material comprises a plurality of protrusions or contact elements, for contacting a tissue site. In still another embodiment, the contact surface comprises a funnel-shaped extension of the first end and the opening comprises the base of the funnel. In a further embodiment of the invention, the funnel-shaped extension comprises a spongiform material. In still a further embodiment of the invention, the first end is detachable from said shaft housing, and one type of first end can be exchanged for another. In another embodiment, attachments are provided for connection with the opening of the first end. For example, in one embodiment, a beveled needle is attached to the opening.
In one embodiment, the second end of the shaft housing is connectable to a syringe, through which the pharmaceutical composition can be delivered to the lumen of the shaft housing. In one embodiment, the syringe is a double-barreled syringe. In still a further embodiment of the invention, delivery of the composition through the syringe is controlled by a motorized element which creates positive or negative pressure within the body of the syringe. In this embodiment, the motorized element can be controlled by a motion of the hand or foot of the operator (e.g., such as by a foot pedal in communication with the motorized element). In still a further embodiment of the invention, the dosage of the pharmaceutical composition is controlled by a processor in communication with an optical system being used to monitor the movement of the contact surface and/or the placement and/or uptake of the pharmaceutical composition.
In another embodiment, kits are provided to facilitate performing the method. In one embodiment, the kit comprises a gene delivery device comprising a contact surface for contacting a tissue; and a pharmaceutical composition comprising a detectable moiety or nucleic acid, or both. In another embodiment of the invention, the gene delivery device comprises a graspable surface for attachment to a contact surface, at least one contact surface for attachment to the graspable surface, and a pharmaceutical composition. In one embodiment of the invention, the gene delivery device is a brush, such as a paintbrush or a toothbrush, or a brush with radially projecting bristles or fibers.
In one embodiment of the invention, the nucleic acids within the pharmaceutical composition are selected from the group consisting of DNA, RNA, anti-sense molecules, triple-helix-forming nucleic acids, aptamers, and ribozymes. In another embodiment of the invention, the nucleic acid is a viral vector, such as an adenoviral vector. In still another embodiment of the invention, the kit includes helper cells or molecules for amplifying the adenoviral vector and for providing a renewable source of the pharmaceutical composition.
In one embodiment of the invention, the gene delivery device is coated with an agent which minimizes adhesion of the pharmaceutical composition to the contact surface and/or the gene delivery device. The use of the anti-adhesive agent is optimized depending on the type of nucleic acid present in the pharmaceutical composition. For example, when the pharmaceutical composition comprises naked DNA, the agent is a DNA repellant such as silane. In an embodiment of the invention where the nucleic acid is encapsulated with viral glycoproteins, the agent is a charged molecule, such as polylysine.
In a further embodiment of the invention, the kit includes a polymerizable compound and a polymerizing agent for enhancing localization of the nucleic acid at the tissue site. In another embodiment of the invention, the polymerizable compound is fibrinogen and the polymerizing agent is thrombin. In a further embodiment of the invention, the pharmaceutical composition comprises detectable moieties, while in a further embodiment of the invention, the kit comprises a solution of detectable moieties which can be added to the pharmaceutical composition.
In one embodiment of the invention, the gene delivery device within the kit comprises a graspable surface having a longitudinal axis and the contact surface is detachable from the graspable surface. In another embodiment, the kit comprises a plurality of contact surfaces, each of which are differently angulated with respect to the longitudinal axis of the grasping element. In still another embodiment, the gene delivery device comprises a shaft housing defining a lumen and having an opening in proximity with the contact surface, the lumen for delivering the pharmaceutical composition to a tissue site being contacted by the contact surface. In a farther embodiment, the contact surface is detachable from the housing. In still a further embodiment, the device comprises a lumen which is turn comprises a first and second channel. The first and second channel can share a common wall. In one embodiment of the invention, the kit comprises a selection of different housings. In another embodiment of the invention, the kit comprises selections of different housings, syringes, adapters, and conduit-tubing for attachment to the gene delivery device.
The use of the device according to the present invention can also be facilitated by providing instructions with the kit. In one embodiment of the invention, the kit comprises instructions including data such as to how to perform the steps of the method. In another embodiment of the invention, the instructions are provided on a CD-ROM or video, or the like.